Ascosphaera apis infects exclusively bee larvae and causes chalkbrood, a lethal fungal disease that results in a sharp reduction in adult bees and colony productivity. However, little is known about the effect of A. apis infestation on the activities of antioxidant enzymes in bee larvae. Here, A. apis spores were purified and used to inoculate Asian honey bee (Apis cerana) larvae, followed by the detection of the host survival rate and an evaluation of the activities of four major antioxidant enzymes. At 6 days after inoculation (dpi) with A. apis spores, obvious symptoms of chalkbrood disease similar to what occurs in Apis mellifera larvae were observed. PCR identification verified the A. apis infection of A. cerana larvae. Additionally, the survival rate of larvae inoculated with A. apis was high at 1–2 dpi, which sharply decreased to 4.16% at 4 dpi and which reached 0% at 5 dpi, whereas that of uninoculated larvae was always high at 1~8 dpi, with an average survival rate of 95.37%, indicating the negative impact of A. apis infection on larval survival. As compared with those in the corresponding uninoculated groups, the superoxide dismutase (SOD) and catalase (CAT) activities in the 5- and 6-day-old larval guts in the A. apis–inoculated groups were significantly decreased (p < 0.05) and the glutathione S-transferase (GST) activity in the 4- and 5-day-old larval guts was significantly increased (p < 0.05), which suggests that the inhibition of SOD and CAT activities and the activation of GST activity in the larval guts was caused by A. apis infestation. In comparison with that in the corresponding uninoculated groups, the polyphenol oxidase (PPO) activity was significantly increased (p < 0.05) in the 5-day-old larval gut but significantly reduced (p < 0.01) in the 6-day-old larval gut, indicating that the PPO activity in the larval guts was first enhanced and then suppressed. Our findings not only unravel the response of A. cerana larvae to A. apis infestation from a biochemical perspective but also offer a valuable insight into the interaction between Asian honey bee larvae and A. apis.

MiRNAs, as a kind of key regulators in gene expression, play vital roles in numerous life activities from cellular proliferation and differentiation to development and immunity. However, little is known about the regulatory manner of miRNAs in the development of Asian honey bee (Apis cerana) guts. Here, on basis of our previously gained high-quality transcriptome data, transcriptome-wide identification of miRNAs in the larval guts of Apis cerana cerana was conducted, followed by investigation of the miRNAs’ differential expression profile during the gut development. In addition to the regulatory network, the potential function of differentially expressed miRNAs (DEmiRNAs) was further analyzed. In total, 330, 351, and 321 miRNAs were identified in the 4-, 5-, and 6-day-old larval guts, respectively; among these, 257 miRNAs were shared, while 38, 51, and 36 ones were specifically expressed. Sequences of six miRNAs were confirmed by stem-loop RT-PCR and Sanger sequencing. Additionally, in the “Ac4 vs. Ac5” comparison group, there were seven up-regulated and eight down-regulated miRNAs; these DEmiRNAs could target 5041 mRNAs, involving a series of GO terms and KEGG pathways associated with growth and development, such as cellular process, cell part, Wnt, and Hippo. Comparatively, four up-regulated and six down-regulated miRNAs detected in the “Ac5 vs. Ac6” comparison group and the targets were associated with diverse development-related terms and pathways, including cell, organelle, Notch and Wnt. Intriguingly, it was noticed that miR-6001-y presented a continuous up-regulation trend across the developmental process of larval guts, implying that miR-6001-y may be a potential essential modulator in the development process of larval guts. Further investigation indicated that 43 targets in the “Ac4 vs. Ac5” comparison group and 31 targets in the “Ac5 vs. Ac6” comparison group were engaged in several crucial development-associated signaling pathways such as Wnt, Hippo, and Notch. Ultimately, the expression trends of five randomly selected DEmiRNAs were verified using RT-qPCR. These results demonstrated that dynamic expression and structural alteration of miRNAs were accompanied by the development of A. c. cerana larval guts, and DEmiRNAs were likely to participate in the modulation of growth as well as development of larval guts by affecting several critical pathways via regulation of the expression of target genes. Our data offer a basis for elucidating the developmental mechanism underlying Asian honey bee larval guts.

Long noncoding RNAs (lncRNAs) are pivotal regulators in gene expression and diverse biological processes, such as immune defense and host–pathogen interactions. However, little is known about the roles of lncRNAs in the response of the Asian honey bee (Apis cerana) to microsporidian infestation. Based on our previously obtained high-quality transcriptome datasets from the midgut tissues of Apis cerana cerana workers at 7 days post inoculation (dpi) and 10 dpi with Nosema ceranae (AcT7 and AcT10 groups) and the corresponding un-inoculated midgut tissues (AcCK7 and AcCK10 groups), the transcriptome-wide identification and structural characterization of lncRNAs were conducted, and the differential expression pattern of lncRNAs was then analyzed, followed by investigation of the regulatory roles of differentially expressed lncRNAs (DElncRNAs) in host response. Here, 2365, 2322, 2487, and 1986 lncRNAs were, respectively, identified in the AcCK7, AcT7, AcCK7, and AcT10 groups. After removing redundant ones, a total of 3496 A. c. cerana lncRNAs were identified, which shared similar structural characteristics with those discovered in other animals and plants, such as shorter exons and introns than mRNAs. Additionally, 79 and 73 DElncRNAs were screened from the workers’ midguts at 7 dpi and 10 dpi, respectively, indicating the alteration of the overall expression pattern of lncRNAs in host midguts after N. ceranae infestation. These DElncRNAs could, respectively, regulate 87 and 73 upstream and downstream genes, involving a suite of functional terms and pathways, such as metabolic process and Hippo signaling pathway. Additionally, 235 and 209 genes co-expressed with DElncRNAs were found to enrich in 29 and 27 terms, as well as 112 and 123 pathways, such as ABC transporters and the cAMP signaling pathway. Further, it was detected that 79 (73) DElncRNAs in the host midguts at 7 (10) dpi could target 321 (313) DEmiRNAs and further target 3631 (3130) DEmRNAs. TCONS_00024312 and XR_001765805.1 were potential precursors for ame-miR-315 and ame-miR-927, while TCONS_00006120 was the putative precursor for both ame-miR-87-1 and ame-miR-87-2. These results together suggested that DElncRNAs are likely to play regulatory roles in the host response to N. ceranae infestation through the regulation of neighboring genes via a cis-acting effect, modulation of co-expressed mRNAs via trans-acting effect, and control of downstream target genes’ expression via competing endogenous RNA networks. Our findings provide a basis for disclosing the mechanism underlying DElncRNA-mediated host N. ceranae response and a new perspective into the interaction between A. c. cerana and N. ceranae.

Non–coding RNA (ncRNA) plays an important role in the regulation of immune responses, growth, and development in plants and animals. Here, the identification, characteristic investigation, and molecular verification of circRNAs in Apis cerana cerana larval guts were conducted, and the expression pattern of larval circRNAs during Ascosphaera apis infection was analyzed. This was followed by exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 3178 circRNAs in the larval guts of A. c. cerana were identified, with a length distribution ranging from 15 nt to 96007 nt. Additionally, 45, 33, and 48 up-regulated circRNAs, as well as 110, 62, and 38 down-regulated circRNAs were identified in the A. apis–inoculated 4–, 5–, and 6–day–old larval guts in comparison with the corresponding uninoculated larval guts. These DEcircRNAs were predicted to target 29, 25, and 18 parental genes, which were relative to 12, 20, and 17 GO terms as well as 144, 114, and 61 KEGG pathways, including five cellular and four humoral immune pathways containing melanization, phagosomes, lysosomes, endocytosis, apoptosis, MAPK, Ras, and Jak–STAT signaling pathways. Furthermore, complex competing endogenous RNA (ceRNA) regulatory networks were detected as being formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The target DEmRNAs were engaged in 36, 47, and 47 GO terms as well as 331, 332, and 331 pathways, including six cellular and six humoral immune-related pathways. In total, nineteen DEcircRNAs, five DEmiRNAs, and three mRNAs were included in the sub-networks relative to three antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). Finally, back–splicing sites within 15 circRNAs and the difference in the 15 DEcircRNAs expression between uninoculated and A.apis–inoculated larval guts were confirmed utilizing molecular methods. These findings not only enrich our understanding of bee host–fungal pathogen interactions, but also lay a foundation for illuminating the mechanism underlying the DEcircRNA–mediated immune defense of A. c. cerana larvae against A. apis invasion.

piRNAs are a class of small non-coding RNAs that play an essential part in genomic defense, as well as in modulation of gene expression and diverse biological processes such as host-pathogen interaction. However, little is known about the expression pattern of the regulatory function of piRNAs in interactions between honey bees and pathogens. In this work, on the basis of our previously obtained high-quality small RNA-seq datasets from western honey bee (Apis mellifera) larval guts, the differential expression profile of piRNAs in A. mellifera larval guts after Ascosphaera apis infestation was analyzed, followed by structural characterization, target prediction, and regulatory network investigation. The potential roles of differentially expressed piRNAs (DEpiRNAs) in regulating host response, especially immune response, were further discussed. In this study, 504, 657, and 587 piRNAs were respectively identified in the 4-, 5-, and 6-day-old larval guts infected by A. apis, with 411 (53.24%) piRNAs shared. The length distribution of these piRNAs ranged from 24 nt to 33 nt and their first base had a C bias, similar to piRNAs discovered in other mammals and insects. Additionally, 96, 103, and 143 DEpiRNAs were detected in the 4-, 5-, and 6-day-old comparison groups; among these, piR-ame-149736, piR-ame-1066173, and piR-ame-1125190 were the most up-regulated, while piR-ame-1202932 was the most down-regulated. There were 68 DEpiRNAs shared between these three comparison groups. The targets of the DEpiRNAs in the three comparison groups were engaged in a suite of crucial functions associated with biological processes, molecular function, and cellular components, including molecular transducer activity, biological regulation, and membrane part. These targets were also relevant to diverse vital pathways such as the phosphatidylinositol signaling system, inositol phosphate metabolism, and Wnt signaling pathway. Further investigation demonstrated that targets of DEpiRNAs were involved in three energy metabolism-related pathways, seven development-associated signaling pathways, and seven immune-relevant pathways, including lysosome and endocytosis, as well as the MAPK and Jak-STAT signaling pathways. The expression trends of five randomly selected DEpiRNAs were verified using a combination of RT-PCR and RT-qPCR. Moreover, the expression levels of six genes targeted by piR-ame-945760 were detected by RT-qPCR, with the results showing that their expression trends were the same as the expression trend of piR-ame-945760, indicative of the positive correlation between piR-ame-945760 and these targets. These results suggest that A.apis infestation increased the overall expression level of piRNAs and altered the expression pattern of piRNAs in A. mellifera larval guts. DEpiRNAs potentially participate in the A. apis response of the host by modulating the expression of target genes associated with energy metabolism and development, as well as cellular and humoral immune response. Our findings not only offer novel insights into A. mellifera larva-A. apis interaction, but also lay the groundwork for clarifying the mechanisms underlying DEpiRNA-regulated larval response.